The usual practice for improving the yield of secondary metabolites produced by a microorganism involves (1) exposing the microorganism to a mutagen and (2) screening random survivors for increased titers of the secondary metabolite. The second step involves individual fermentation of each surviving isolate. This is a very expensive, time-consuming operation.
Mycophenolic acid (MPA) is a compound with several useful biological activities. It is an antiviral and antitumor agent [K. Ando et al., J. Antibiot. (Tokyo) 21, 649-652 (1968) and R. H. Williams et al., J. Antibiot. (Tokyo) 21, 463-464 (1968)]. Mycophenolic acid is also an antifungal and an antibacterial agent [K. Gilliver, Ann. Bot. (London) 10, 271-282 (1946) and E. Abraham, Biochem. J. 39, 398-408 (1945)]. Mycophenolic acid has also been reported to be useful in the treatment of psoriasis [I. S. Johnson, Chem. Abstr. 77:92853 (1972)].
Mycophenolic acid is produced by many species of Penicillium, e.g., P. brevi-compactum, P. stoloniferum, P. scabrum, P. nagemi, P. szaferi, P. patus-mei, P. griscobrunneum, and P. viridicatum [P. W. Clutterbuck et al., Biochem. J. 26, 1442-1458 (1932)]. In fact, mycophenolic acid was initially isolated from a culture of Penicillium [B. Gosio, Riv. Igiene Sanita Pub. Ann. 7, 825-869 (1896)].
The structure of mycophenolic acid was determined chiefly by Raistrick et al. [J. H. Birkinshaw, H. Raistrick, and D. J. Ross, Biochem. J. 50, 630-634 (1952)]. Mycophenolic acid was found to have the following structure: ##STR1##
In the biosynthesis of mycophenolic acid by Penicillium species, it has been shown that both the phenolic nucleus and the seven-membered side chain of MPA are formed from acetate units (A. J. Birch et al., J. Chem. Soc. 1958, 369-375). The side chain of MPA arises by introduction of a C.sub.15 terpene, presumably farnesylpyrophosphate (FPP), followed by oxidative fission at the double bond of the FPP moiety [L. Canonica et al., J. Chem. Soc. Perkins Trans. I. 21, 2639-2653 (1972)]. FPP also serves as a precursor of sterols (E. Heftman et al., "Biochemistry of Steroids," Reinhold Publishing Corp, New York, 1960, p. 231). Ergosterol is an important constituent of fungal membranes (D. Gottlieb, `Functions of Sterols in Fungi` in "Morphological and Biochemical Events in Plant Parasite Interaction," S. Akai, Ed., Mochizuki Publishing Co., Omiya, Japan, 1971).
Polyene antibiotics such as, for example, nystatin, filipin, and amphotericin B are known to exert a toxic effect on fungi by binding with the ergosterol in fungal membranes [J. M. T. Hamilton-Miller, Adv. Appl. Microbiol. 17, 109-134 (1974)]. The primary mechanism of resistance to polyene antibiotics develops in fungi by genetic interruption of the biosynthesis of ergosterol, producing sterols with less affinity to polyene antibiotics. These sterols then produce fungal membranes with altered permeability characteristics. Another possible class of polyene-resistant mutants includes those which overproduce ergosterol, thereby detoxifying the antibiotics before they reach the membrane [W. A. Zygmunt and P. A. Tavormina, Appl. Microbiol. 14, 865-869 (1966)]. Two classes of polyene-resistant mutants might overproduce mycophenolic acid by overproducing FPP: (1) those in which the synthesis of FPP is normal, but synthesis of ergosterol is blocked after FPP, and (2) those which overproduce both FPP and ergosterol.